Studies of virulence factors of Bacillus anthracis, the causative agent of anthrax, have mainly focused on plasmid-encoded capsule as well as lethal and edema toxins. Very few studies have been reported on pore-forming hemolysins. A primary reason for the limited studies is that B. anthracis is generally accepted as a nonhemolytic pathogen, although hemolysin genes are present in the genome. However, recent studies have shown that B. anthracis can produce hemolysin under certain conditions. Another nonhemolytic sporeformer Bacillus subtilis also carries putative hemolysin genes. The long-term objectives of the research plan are to investigate whether hemolysin plays a role in pathogenicity of B. anthracis such as successful escape from macrophage into extracellular milieu. The proposed studies will aim at understanding how virulence factors such as hemolysins are induced in response to environmental changes and as a result of a single mutation in regulatory genes. The activation of 'latent' virulence genes could have a strong impact on various bacterial infections. The specific aims of the proposal are: 1) To identify regulatory mutation(s) that activate ?-hemolysin gene(s) in B. subtilis by genome mapping and DNA sequence analysis. 2) To characterize the Induction mechanism of the ?-hemolysin gene by the regulatory mutation and by oxygen limitation using a transcriptional lacZ fusion to the promoter of the hemolysin gene. 3) To investigate the stimulatory effect of nitrate on anaerobic expression of ?-hemolysin gene in B. subtilis and on aerobic/anaerobic expression in B. anthracis. The hypothesis that nitrate-dependent induction requires nitrate reductase activity will be examined by using a B. subtilis nitrate reductase mutant and measuring nitrate reductase activity of aerobic B. anthracis cultures. B. anthracis gene(s) encoding putative transcriptional regulator(s) of the CRP-family will be introduced into B. subtilis to determine whether the putative regulator is able to activate expression of the respiratory nitrate reductase operon under aerobic conditions, and thus leading to the aerobic induction of ?-hemolysin in B. subtilis. [unreadable] [unreadable] [unreadable]